CN108802259B

CN108802259B - Detection method of bupleurum chinense

Info

Publication number: CN108802259B
Application number: CN201810597316.1A
Authority: CN
Inventors: 康兴东; 杨明; 梁佳; 舒积成; 杜剑松; 伍振峰; 周朝忠; 张金良
Original assignee: JIANGXI PUZHENG PHARMACEUTICAL CO Ltd; Jiangxi University of Traditional Chinese Medicine
Current assignee: JIANGXI PUZHENG PHARMACEUTICAL CO Ltd; Jiangxi University of Traditional Chinese Medicine
Priority date: 2018-06-11
Filing date: 2018-06-11
Publication date: 2020-07-07
Anticipated expiration: 2038-06-11
Also published as: CN108802259A

Abstract

The invention relates to a detection method of bupleurum chinense, and belongs to the technical field of detection of traditional Chinese medicinal materials. The invention provides a novel method for detecting bupleurum chinense. The method uses kaerophyllin as reference, adopts thin layer chromatography for qualitative identification, has good reproducibility, good separation degree, simple operation and low cost, and enhances quality guarantee of bupleuri radix.

Description

Detection method of bupleurum chinense

The technical field is as follows:

the invention relates to a detection method of bupleurum chinense, and belongs to the technical field of detection of traditional Chinese medicinal materials.

Background art:

bupleurum longiradiatum Wall ex DC, belonging to Bupleurum genus of Umbelliferae family, has effects of dispelling pathogenic wind, removing heat, dispersing stagnated liver qi, and invigorating yang, and can be used for treating common cold, fever, alternating cold and heat, malaria, chest and rib distending pain, menoxenia, uterine prolapse, proctoptosis, etc. The bupleurum root is mainly distributed in the provinces of Sichuan, Hubei, Yunnan, Guizhou and the like in China, and is a local article for bupleurum root medicinal materials in southwest of China.

At present, the detection technology of the bupleurum chinense is less, and particularly, the thin-layer identification method is more insufficient. A thin-layer identification method for bupleuri radix in Hunan province by using saikoside a and saikoside d as reference substances in Chinese medicinal material standard (2009 edition) is provided. Plum 26107. The method for identifying saponin components in bupleuri radix by thin layer chromatography with saikoside a and saikoside d as reference substances and the method for identifying flavonoid components in bupleuri radix by thin layer chromatography with rutin, quercetin, kaempferol and isorhamnetin as reference substances are respectively established. The existing thin-layer identification method for the bupleurum bambusoides is mainly used for identifying saponin components or flavonoid components in the bupleurum bambusoides, a method for identifying characteristic lignanoid components in the bupleurum bambusoides is lacked, the covered chemical components are insufficient, and the quality of the medicinal materials of the bupleurum bambusoides cannot be effectively guaranteed.

The bupleuri radix has various chemical components and structure types, and mainly contains saponins, flavonoids, lignanoids, coumarins, volatile oil, etc.

The invention content is as follows:

aiming at the problems, the invention aims to provide a novel method for detecting bupleurum chinense Pursh, which is simple and convenient to operate,

the detection method of the bupleurum chinense nakai takes lignans as a reference and adopts thin-layer chromatography for qualitative identification and analysis.

The detection method of the bupleurum chinense of the invention comprises the following steps: qualitative identification by thin layer chromatography with kaerophyllin as control comprises the following steps:

a. preparation of control solutions: adding methanol into kaerophyllin reference substance to obtain solution containing 0.1mg per 1ml as reference substance solution;

b. preparation of a test solution: taking 1g of bupleurum powder of bamboo leaves, adding 10-30 ml of methanol, carrying out ultrasonic treatment for 20-40 min, filtering, evaporating filtrate to dryness, adding 10-20 ml of methanol into residues for dissolving, extracting 1 time by 10-20 ml of petroleum ether, taking methanol parts, evaporating to dryness, adding 10-20 ml of water into residues for dissolving, extracting 1 time by 10-20 ml of n-hexane, taking n-hexane parts, evaporating to dryness, adding 1ml of methanol for dissolving, and taking the n-hexane parts as a sample solution;

c. identification by thin layer chromatography: respectively dispensing 3-7 μ l of the reference substance solution and 3-7 μ l of the test substance solution on the same silica gel GF254 thin-layer plate, developing with petroleum ether, acetone and ammonia water at a ratio of 30-50: 15-25: 0.5-1.5 as developing agent, taking out, air drying, and placing under an ultraviolet lamp at 254nm for inspection, wherein spots with the same color are respectively displayed on the positions corresponding to the reference substance chromatogram in the test substance chromatogram;

wherein steps a and b are not in sequence.

Thin-layer chromatography identification research on bamboo leaf and radix bupleuri

Preparation of a test solution: the extraction solvents such as methanol, 90% methanol, 80% methanol, 70% methanol, anhydrous ethanol, 95% ethanol, and 80% ethanol were examined, indicating that the methanol extraction was better. The ultrasonic treatment for 30 minutes was found to be effective when the extraction method, such as ultrasonic treatment (20 minutes, 30 minutes, 40 minutes) and reflux (1 hour, 2 hours, 3 hours), was examined.

Direct sample application after methanol extraction, normal hexane extraction and sample application after methanol extraction, petroleum ether impurity removal and normal hexane extraction and sample application after methanol extraction are examined, and the results show that direct sample application after methanol extraction and normal hexane extraction and sample application after methanol extraction are simple in operation and capable of achieving the identification effect, but the impurities are more, and the interference to the background is large. And after the petroleum ether is subjected to impurity removal after the methanol extraction and then is subjected to n-hexane extraction, the sample application can achieve a good separation effect, and the background interference is small.

Comparison products: the kaerophyllin is extracted and separated from bupleurum chinense, and the structure is identified by spectrum and chromatography, and the purity is more than or equal to 98 percent.

As one skilled in the art knows, when performing thin layer chromatography identification, chemical components, stationary phase and developing solvent are interactive, and different chemical component substances need to be separated and identified by finding appropriate stationary phase and developing solvent. The existing thin-layer chromatography identification method for bupleurum chinense DC is used for separating and identifying chemical component substance groups such as saponins, flavonoids and the like; the invention firstly uses lignans as a reference to carry out thin-layer identification on the bupleurum chinense, and has creativity. Therefore, the invention researches a specific stationary phase and a developing agent suitable for the thin-layer chromatography separation of the bupleurum chinense and lignans components. Different thin-layer plates such as self-made GF254 plates, prefabricated GF254 plates and prefabricated GF254 efficient plates have good separation effects, but the self-made plates are not as attractive as the prefabricated plates, and the prefabricated GF254 plates are selected.

Developing agent: various development systems such as petroleum ether-ethyl acetate, petroleum ether-acetone, n-hexane-ethyl acetate, petroleum ether-acetone-ammonia water and the like are investigated. The optimal development system is a development system with the ratio of petroleum ether, acetone and ammonia water being 40:20:1, the separation effect is good, the spots are clear, and the impurities are not interfered.

Humidity effect: the influence of different humidity conditions such as 22%, 42%, 72% and the like on the unfolding effect is examined, when the relative humidity is 72%, spots are fuzzy, and normal humidity is selected.

The invention provides a new method for detecting bupleurum chinense, which has the advantages of high accuracy, good reproducibility, good separation degree, simple operation, low cost, small toxicity of used reagents and small dosage, and enhances the quality guarantee of the bupleurum chinense medicinal material.

Drawings

FIG. 1 investigation of different lamella plates

FIG. 2 examination of different developing Agents

FIG. 3 differential humidity investigation

FIG. 4 thin layer chromatogram of Bupleurum scorzonerifolium of different batches

Detailed Description

To better explain the practice of the present invention, examples of the following assay methods are provided. These examples are merely illustrative and do not limit the scope of the invention. The invention is further illustrated by the following exemplary embodiments.

Example 1:

a kaerophyllin control was added with methanol to prepare a solution containing 0.1mg of methanol per 1ml, and the solution was used as a control solution. Taking 1g of Bupleurum falcatum powder, adding 10ml of methanol, carrying out ultrasonic treatment for 20min, filtering, drying the filtrate by distillation, adding 10ml of methanol into the residue for dissolving, extracting with 20ml of petroleum ether for 1 time, taking the methanol part, drying by distillation, adding 10ml of water into the residue for dissolving, extracting with 20ml of n-hexane for 1 time, taking the n-hexane part, drying by distillation, adding 1ml of methanol for dissolving, and taking the obtained solution as a sample solution. Taking 3 μ l of the above control solution and 3 μ l of the test solution according to thin layer chromatography (0502 of the national pharmacopoeia 2015 edition), respectively dropping on the same silica gel GF254 thin layer plate, developing with petroleum ether, acetone and ammonia water at a ratio of 50:15:1.5 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 254 nm. Spots of the same color appear in the chromatogram of the test solution at the corresponding positions in the chromatogram of the control solution.

Example 2:

a kaerophyllin control was added with methanol to prepare a solution containing 0.1mg of methanol per 1ml, and the solution was used as a control solution. Taking 1g of Bupleurum falcatum Thunb powder, adding 30ml of methanol, performing ultrasonic treatment for 40min, filtering, evaporating filtrate to dryness, dissolving residue with 20ml of methanol, extracting with 10ml of petroleum ether for 1 time, taking methanol part, evaporating to dryness, dissolving residue with 20ml of water, extracting with 10ml of n-hexane for 1 time, taking n-hexane part, evaporating to dryness, and dissolving with 1ml of methanol to obtain a sample solution. Taking 7 μ l of the above control solution and 7 μ l of the test solution according to thin layer chromatography (0502 of the national pharmacopoeia 2015 edition), respectively dropping on the same silica gel GF254 thin layer plate, developing with petroleum ether, acetone, ammonia water (30: 25: 0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 254 nm. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.

Example 3:

a kaerophyllin control was added with methanol to prepare a solution containing 0.1mg of methanol per 1ml, and the solution was used as a control solution. Taking 1g of Bupleurum falcatum Thunb powder, adding 20ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving residue with 15ml of methanol, extracting with 15ml of petroleum ether for 1 time, taking methanol part, evaporating to dryness, dissolving residue with 15ml of water, extracting with 15ml of n-hexane for 1 time, taking n-hexane part, evaporating to dryness, and dissolving with 1ml of methanol to obtain a sample solution. Taking 5 μ l of the above control solution and 5 μ l of the test solution according to thin layer chromatography (0502 of the national pharmacopoeia 2015 edition), respectively dropping on the same silica gel GF254 thin layer plate, developing with petroleum ether, acetone and ammonia water (40: 20: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 254 nm. Spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.

Example 4:

for different thin layer plate, see FIG. 1

For the following steps: kaerophyllin; 1-4: radix bupleuri of different batches

A: self-platemaking

B: precast slab (Qingdao ocean chemical Co., Ltd.)

C: high efficiency precast slab (Yantai river friend silica gel development Co., Ltd.)

Example 5:

examination of the different developing agents, see FIG. 2

For the following steps: kaerophyllin; 1-3: radix bupleuri of different batches

A: petroleum ether; ethyl acetate 3:2

B: petroleum ether acetone 4:1.5

C: n-Hexane Ethyl acetate 3:2

D: petroleum ether, acetone and ammonia water 40:20:1

Example 6:

for different humidity examinations, see FIG. 3

For the following steps: kaerophyllin; 1-4: radix bupleuri of different batches

Example 7:

the thin layer chromatogram of bupleuri radix in different batches is shown in FIG. 4

For the following steps: kaerophyllin; 1-15: radix bupleuri of different batches

Claims

1. A detection method of bupleurum chinense nakai is characterized in that:

qualitative identification with kaerophyllin reference by thin layer chromatography comprises the following steps:

wherein the steps a and b have no sequence.

2. The method for detecting bupleurum longiradiatum as claimed in claim 1, wherein:

b. preparation of a test solution: taking 1g of Bupleurum falcatum Thunb powder, adding 20ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving residue with 15ml of methanol, extracting with 15ml of petroleum ether for 1 time, taking methanol part, evaporating to dryness, dissolving residue with 15ml of water, extracting with 15ml of n-hexane for 1 time, taking n-hexane part, evaporating to dryness, and dissolving with 1ml of methanol to obtain a sample solution;

c. identification by thin layer chromatography: respectively dispensing 5 μ l of the reference solution and 5 μ l of the test solution on the same silica gel GF254 thin-layer plate, developing with petroleum ether, acetone and ammonia water at a ratio of 40:20:1 as developing agent, taking out, air drying, and inspecting under an ultraviolet lamp at 254nm to obtain spots with the same color on the corresponding positions of the control chromatogram in the test chromatogram;

wherein the steps a and b have no sequence.